Rational antigen engineering and mucosal delivery design for next-generation RSV vaccines

Respiratory syncytial virus (RSV) prefusion F (preF) vaccines have transformed adult prophylaxis, yet unmet needs in antigen stability, pediatric safety, and mucosal protection persist. Here, we develop an integrated structure-guided RSV vaccine design platform that couples allosteric stabilization, epitope-focused immunogen engineering, and route-specific mRNA delivery for systemic and mucosal immune activations. By mapping prefusion F “breathing” motions and applying a ThermoNet-and Rosetta-guided screening funnel, we identified R296, a stabilized prefusion F immunogen that reinforces the α1–α5 hinge and interprotomer interfaces while preserving key neutralizing epitopes. Cryo-EM confirmed that R296 retains a native-like prefusion architecture. And mRNA-LNP vaccination elicited potent, durable, and broadly protective neutralizing responses in mice, rats, and cotton rats, with clearance of detectable infectious virus and no evidence of Th2-skewed enhanced respiratory disease. To address pediatric safety, we designed a stalkless nanoparticle immunogen, Head38-50AB-3, which enriches high-potency apical epitopes while excluding stalk regions associated with low-potency or non-protective responses, conferring protection without VAERD-like pathology. Finally, we engineered an intranasal-delivered LNP that enables intranasal R296 mRNA delivery, inducing systemic neutralization together with robust nasal and bronchoalveolar secretory IgA (sIgA). R296 has now advanced to Phase 1 clinical trials. These results establish a modular framework for next-generation RSV vaccines.